Different methods and devices are known for counting microorganism colonies in, for example, petri dishes. Manual counting of colonies by trained laboratory personnel is well-known. This method has many disadvantages. They include the costs associated with the use of skilled technicians to perform the time-consuming chore of manual counting as well as the limited accuracy in the counts achieved.
Automated counting systems are also known. They can be separated into two basic categories. The first category includes those systems employing cameras or video equipment in conjunction with hard-wired circuits or digital computers to count the number of colonies in a petri dish. Examples of such systems are described in EP Publication No. 0 301 600; U.S. Pat. No. 3,811,036 to Perry; and French Publication No. 2 602 074.
Video-based systems suffer from a number of disadvantages. The primary disadvantage is the expensive and sophisticated equipment used in such systems to process the raw pixel image produced by the video cameras. To avoid multiple counting of the same colonies such systems typically include processing-intensive labelling schemes requiring relatively powerful computer systems to accurately count of the number of colonies in an acceptable amount of time.
An additional disadvantage is that many of these video-based systems require that the petri dishes be illuminated through their bottom surface which requires a substrate which is light permeable to ensure accurate counting. The illumination is typically required because of the thickness of the agar used in petri dishes results in colonies growing on the surface of the agar as well as in the middle and on the bottom of the agar. Surface illumination only would result in undercounting of the colonies in the middle and on the bottom of the agar. In other systems, such as that described in EP 0 301 600, the absorbance and transmission of light is used to detect colonies.
The growth of colonies throughout the vertical thickness of the medium is not a particular problem for lasers of the disposable microorganism culturing devices such as PETRIFILM.TM., manufactured by 3M Company. Such devices have a very thin layer of growth medium making all colonies visible with surface illumination. The substrate is not, however, sufficiently permeable to light for use with many of the known automated counting systems.
In addition to the cost and complexity of the hardware configurations of known automated video counting systems, the object counting algorithms used with systems employing digitization of the images also suffer from disadvantages.
A simple Euler number can be used to identify objects in a raster-scanned image and can be used with only a single pass through the image, but detects only 4-connected objects. This can give spurious results when 8-connected images are present in the scanned image.
At the opposite extreme, a full-connected component analysis detects all objects, whether they are 4-connected or 8-connected. That type of analysis, however, involves complicated-labeling and tagging operations which can require multiple passes through the image, as well as significantly more complex and costly hardware.
The second category of automated counting systems typically uses an array of photodetectors and hard-wired circuitry to perform the counting process. As with most of the video-based systems, the counting systems using photodetectors are also limited by the requirement that the petri dish be illuminated through its substrate to produce an accurate count. As a result, the substrate on which the colonies are contained must be light permeable, which is a particular problem with disposable culturing devices such as PETRIFILM.TM..
Examples of such systems are disclosed in U.S. Pat. No. 3,493,772 to Daughters II et al. as well as U.S. Pat. No. 3,736,432 to Sweet. The Daughters' device includes a light source located below the petri dish and uses the colonies as lenses to focus the light on a linear array of photodetectors. The Sweet device employs a light source below the petri dish which uses the light absorbency of the bacterial colonies to produce dark spots on a linear photo detector. Both systems use dedicated circuitry to perform the counting process.
The Daughters and Sweet devices both use a linear array of photodetectors and rely on voltage changes to inhibit counting by neighboring photodetectors, thereby avoiding multiple counting of individual colonies. That circuitry can cause errors in counting because colonies which may or may not be opaque or light permeable may not be counted with a given system. Other errors can occur in the counting of tree-shaped or other complex shaped colonies.